羊源多杀性巴氏杆菌toxA-N基因的克隆、原核表达及生物信息学分析  

Cloning,Prokaryotic Expression and Bioinformatics Analysis of toxA-N Gene of Pasteurella multocidain Goat

作　　者：黄海峰 王成强 张振兴 李宝宝 郑义盈 安琪 张萌萌 章泸尹 朱姝 曹瑞勇 杨小健 聂鑫 杜丽 王凤阳 HUANG Haifeng;WANG Chengqiang;ZHANG Zhenxing;LI Baobao;ZHENG Yiying;AN Qi;ZHANG Mengmeng;ZHANG Luyin;ZHU Shu;CAORuiyong;YANG Xiaojian;NIE Xin;DU Li;WANG Fengyang(Key Laboratory of Animal Genetic Engineering of Haikou City ,Key Laboratory of Tropical Animal Reproduction & Breeding and Kpidemic Disease Research of Hainan Province ,College of Animal Science and Technology,College of Tropical Agriculture and Forestry,Hainan University,Haikou 570228 ,China)

机构地区：[1]海南大学热带农林学院动物科技学院,海南省热带动物繁育与疫病研究重点实验室,海口市动物基因工程重点实验室,海口570228

出　　处：《中国畜牧兽医》2018年第12期3337-3345,共9页China Animal Husbandry & Veterinary Medicine

基　　金：海南省重大科技计划项目(ZDKJ2016017-01);国家肉羊产业技术体系(CARS-38);中央引导地方科技发展专项资金项目(ZY2017HN07)

摘　　要：试验旨在对羊源多杀性巴氏杆菌toxA-N基因进行克隆、原核表达及纯化,并对表达蛋白toxA-N进行生物信息学分析,为探索羊源多杀性巴氏杆菌毒素基因的相关特性提供参考依据。以羊源多杀性巴氏杆菌基因组为模板,参考GenBank中公布的多杀性巴氏杆菌HN06中toxA基因序列(登录号:CP003313.1)设计引物,通过PCR技术扩增出目的片段,构建重组质粒pET28a(+)-toxA-N,转化E.coli BL21(DE3)感受态细胞,IPTG诱导表达后,经考马斯亮蓝染色及Western blotting鉴定,纯化蛋白并运用生物信息学软件对表达蛋白进行特性分析。结果显示,试验成功扩增出大小为1 515bp的toxA-N基因片段,经BamHⅠ和NotⅠ双酶切得到大小约为5 369和1 515bp两条片段,表明成功构建了pET28a(+)-toxA-N重组质粒,IPTG诱导表达的菌株经考马斯亮蓝染色及Western blotting鉴定后,成功表达出大小约为60ku的toxA-N蛋白;生物信息学分析表明,toxA-N蛋白为包涵体,其分子式为C2635H4002N664O797S17,原子总个数为8 115,消光系数为84 480,不稳定指数为43.50,亲水性平均值为-0.381。在toxA-N蛋白二级结构中,α-螺旋、β-折叠、延伸链和无规则卷曲分别占53.47%、2.77%、11.28%和32.48%,与三级结构预测结果一致。本试验通过对toxA-N基因的初步研究,揭示了多杀性巴氏杆菌毒素的相关特性,对家畜的疾病预防、诊断、治疗具有重要意义。This study was aimed to clone,prokaryotically express and purify toxA-N gene of Pasteurella multocida in goat,and analyze the expressed protein toxA-N by bioinformatics,which provided a reference for exploring the relevant characteristics of the Pasteurella multocida toxingene in goat.The genome of the above-mentioned bacteria was used as a template,and the primer was designed with reference to the Pasteurella multocida HN06 toxAgene sequence published in GenBank(accession No.:CP003313.1),and the target fragment was amplified by PCR.The recombinant plasmid pET28 a(+)-toxA-N was constructed and then transferred to E.coli BL21(DE3)competent cells.After inducted by IPTG,the expressed proteins were characterized by Coomassie blue staining,Western blotting,protein purification and bioinformatics softwares.The results showed that the 1 515 bp gene fragment was successfully amplified,and the two fragments of 5 369 and 1 515 bp were digested by BamH Ⅰ and Not Ⅰ,indicating that the recombinant plasmid pET28 a(+)-toxA-N was successfully constructed.The Coomassie blue staining and Western blotting results showed that a size of about 60 ku toxA-N protein was successfully expressed by IPTG induction.Bioinformatics analysis results showed that toxA-N protein was an inclusion body with a molecular formula of C2635 H4002 N664 O797 S17,the total number of atoms was8 115,the extinction coefficient was 84 480,the instability index was 43.50,and the average hydrophilicity was-0.381.In the secondary structure of toxA-N protein,α-helix,β-turn,extended chain and random coil accounted for 53.47%,2.77%,11.28% and 32.48%,respectively,which were consistent with the prediction results of the tertiary structure.In summary,this study revealed the relevant properties of the Pasteurellamultocidatoxin by preliminary study of toxA-N gene,which was of great significance for disease prevention,diagnosis and treatment of livestock.

关 键 词：羊源多杀性巴氏杆菌 toxA-N基因 亚克隆 生物信息学分析 

分 类 号：S852.[农业科学—基础兽医学]